RESUMEN
Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrin into cyanide and the corresponding aldehyde or ketone. Moreover, they catalyze the synthesis of cyanohydrin in the reverse reaction, utilized in industry for preparation of enantiomeric pure pharmaceutical ingredients and fine chemicals. We discovered a new HNL from the cyanogenic millipede, Chamberlinius hualienensis. The enzyme displays several features including a new primary structure, high stability, and the highest specific activity in (R)-mandelonitrile ((R)-MAN) synthesis (7420 U·mg-1 ) among the reported HNLs. In this study, we elucidated the crystal structure and reaction mechanism of natural ChuaHNL in ligand-free form and its complexes with acetate, cyanide ion, and inhibitors (thiocyanate or iodoacetate) at 1.6, 1.5, 2.1, 1.55, and 1.55 Å resolutions, respectively. The structure of ChuaHNL revealed that it belongs to the lipocalin superfamily, despite low amino acid sequence identity. The docking model of (R)-MAN with ChuaHNL suggested that the hydroxyl group forms hydrogen bonds with R38 and K117, and the nitrile group forms hydrogen bonds with R38 and Y103. The mutational analysis showed the importance of these residues in the enzymatic reaction. From these results, we propose that K117 acts as a base to abstract a proton from the hydroxyl group of cyanohydrins and R38 acts as an acid to donate a proton to the cyanide ion during the cleavage reaction of cyanohydrins. The reverse mechanism would occur during the cyanohydrin synthesis. (Photo: Dr. Yuko Ishida) DATABASES: Structural data are available in PDB database under the accession numbers 6JHC, 6KFA, 6KFB, 6KFC, and 6KFD.
Asunto(s)
Acetonitrilos/química , Aldehído-Liasas/química , Proteínas de Artrópodos/química , Artrópodos/química , Lipocalinas/química , Acetonitrilos/metabolismo , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Artrópodos/enzimología , Sitios de Unión , Biocatálisis , Clonación Molecular , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Ácido Yodoacético/química , Ácido Yodoacético/metabolismo , Cinética , Lipocalinas/genética , Lipocalinas/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Tiocianatos/química , Tiocianatos/metabolismoRESUMEN
The enzyme responsible for the enantioselective production of (S)-1,1,1-trifluoro-2-propanol ((S)-TFP) from 1,1,1-trifluoroacetone (TFA) has been identified in Ogataea polymorpha NBRC 0799. We purified two carbonyl reductases, OpCRD-A and OpCRD-B from this strain, and revealed their characteristics. Both enzymes were specific to NADH, but the following characteristics were different: The molecular mass of subunit OpCRD-A was 40 kDa and that of OpCRD-B was 43 kDa. Amino acid sequences of both enzymes were only 21% identical. OpCRD-B contained 4 mol of zinc per mole of enzyme, but OpCRD-A did not. The optimal pH, temperature, pH stability, thermostability, and inhibitor specificity were also remarkably different. With regard to substrate specificity, both enzymes exhibited high reductase activity toward a wide variety of ketones, aldehydes and fluoroketones, and dehydrogenase activity toward 2-propanol and 2-butanol. The reductase activity was much higher than the dehydrogenase activity at acidic pH. OpCRD-A enantioselectively produced (S)-TFP from TFA, but OpCRD-B preferentially produced (R)-TFP. Thus, we concluded that OpCRD-A plays the main role in the production of (S)-TFP by a reaction of O. polymorpha NBRC 0799 cells and that OpCRD-A has great potential for efficient production of (S)-TFP, as it is an S-specific enzyme and does not catalyze the dehydrogenation of (S)-TFP.